1. Field of the Invention
This invention pertains to the systemic administration of an active agent, a recombinant gene comprising an adenovirus (Ad) which contains an osteocalcin promoter (OC) which drives the expression of thymidine kinase (TK). The agent itself is fully disclosed in the parent application. This invention pertains to the discovery that Ad-OC-TK may be administered systemically, both to treat tumors, and to treat certain benign conditions such as benign prostatic hypertrophy and certain forms of arteriosclerosis.
2. Background of Related Work
Toxic gene therapy for the treatment of cancer continues to gain prominence in basic research, but remains limited in clinical application because of an inability to deliver the toxic gene to the tumor cells with specificity. Many vectors (e.g. retroviruses, retroviral producing cells, adenoviruses, liposomes, and others) can deliver genes (therapeutic or toxic) to target cells. Localized delivery and restricted gene expression to the primary tumor have been accomplished via direct injection of therapeutic viruses in animal models1-4 and clinical trails.5,6 This approach is not feasible for the treatment of metastatic disease because of the presence of multiple lesions that would each require separate injection and manipulation. Therefore, alternative approaches to the treatment of metastatic disease with gene therapy must be developed.
Systemic delivery of therapeutic genes is attractive for targeting metastatic disease, pulmonary metastases in particular. Because the pulmonary vascular system would be the first encountered, the adenovirus would be trapped in the lung parenchyma, allowing for higher infectivity. Lesoon-Wood et al,.7 reported the systemic delivery of wild type p53 complexed with liposomes, targeting the p53 mutated breast cancer cell line (MDA-MB435), inhibiting primary tumor growth by 60%, and decreasing pulmonary metastases in nude mice. Vile et al.8 demonstrated inhibition of B-16 melanoma pulmonary metastases in syngeneic immunocompetent mice by a systemic delivery of retrovirus using a tyrosinase promoter to drive the expression of the toxic gene thymidine kinase (TK) gene.
Compared to liposome or retrovirus, adenovirus has several advantages in a systemic delivery strategy, such as its high infectivity in vivo and production techniques that can achieve high viral titers. However, Brand et al.9 recently reported that systemic administration of adenovirus containing TK under the control of a universal promoter (CMV) supplemented with ganciclovir treatment induced severe hepatotoxic effects. This study suggested that restriction of toxic gene (TK) expression by tissue specific promoter may be necessary prior to the consideration of systemic adenoviral vector delivery. Moreover, the tissue-specific promoter should limit the toxic gene expression in normal tissues so it can be applied in higher doses than the universal promoter-based toxic gene therapy for more effective treatment of metastatic diseases.
To study the potential therapeutic efficacy of systemic cancer gene therapy for the treatment of pulmonary metastases, osteosarcoma is an attractive model because a significant number of these patients eventually develop lung metastasis. Initially, surgical resection of the primary lesion and adjunctive chemotherapy are the mainstay of today""s therapy. For the 20% that present with metastatic disease, 80% will require additional therapy for relapse; while of the 80% that present with local disease, 35% will require additional therapy for relapse after surgery and adjunctive chemotherapy.10 Therefore, 44% of patients diagnosed with osteosarcoma will fail conventional first line therapy. Patients developing recurrent disease usually have a poor prognosis, dying within one year of the development of metastatic disease.11-14 New therapeutic approaches that can be applied either separately or in conjunction with current modalities in treating osteosarcoma pulmonary metastases are needed.
The osteocalcin promoter (OC) has been shown to be highly effective in directing the transcription of reporter genes in both rat and human osteosarcoma cell lines.4,15 In parent application U.S. Ser. No. 08/785,088 and concurrent publications it was shown that a recombinant adenovirus containing TK gene under the control of OC promoter, when supplemented with a prodrug ACV, could suppress osteosarcoma growth through intralesional injection in both rat and human osteosarcoma models.1,4 
Osteosarcoma, a bone cancer occurring primarily in teenagers and young adults, affects approximately 2100 individuals yearly in the United States (Boring, C. C., Squires, T. S., Tong, T., and Montgomery. S. Cancer statistics, 1994, CA Cancer J. Clin., 44; 7-26, 1994). This malignancy accounts for as many as 5% of all childhood malignancies and 60% of all malignant childhood bone tumors (Hudson, M., Jaffe, M. R., and Jaffe, N. Pediatric osteosarcoma: therapeutic strategies, results, and prognostic actors derived from a 10 10-year experience. J. Clin. Oncol., 8: 1988-1997, 1990). Despite radical surgical resection of the primary tumor and aggressive adjuvant chemotherapy, the overall 2-year metastasis-free survival rate approaches only 66%. More than 30% of patients with this disease develop lung metastasis within the first year (Link, M. P., Goorin, A. M., Mixer, A. W., Link, M. P., Goorin, A. M., Miser, A. W., Green, A. A., Pratt, C. H., Belasco, J. B., Pritchard, J., Malpas, J. S., Baker, A. R., Kirkpatrick, J. A., Ayala, A. O., Schuster, J. J., Abelson, H. T., Simone, J. V., and Vietti, T. J. The effect of adjuvant chemotherapy on relapse-free survival in patients with osteosarcoma of the extremity. N. Engl. J. Med, 314: 1600-1602, 1991. Goorin, A. M., Perez-Atayde, A., Gebbhardt, M., et al. Weekly high-dose methotrexate and doxorubicin for osteosarcoma: the Dunn-Farber Cancer Institute/The Children""s Hospital-Study III. J. Clin. Oncol., 5: 1178-1184, 1987). The survival rate among those affected with osteosarcoma has not changed significantly over the past 10 years, despite changes in adjuvant chemotherapy, Kane, M. J. Chemotherapy of advanced soft tissue and osteosarcoma. Semin. Oncol., 16:297-304, 1989.
The concept of delivery and expression of therapeutic toxic genes to tumor cells through the use of tissue-specific promoters has been well recognized. This approach could decrease the toxic effect of therapeutic genes on neighboring normal cells when virus-mediated gene delivery results in the infection of the normal cells. Examples include the uses of the albumin or xcex1-fetoprotein promoter to target hepatoma cells (Kuriyama, S., Yoshikawa, M., Ishizaka, S., Taujli, T., Ikenaka, K., Kagawa, T., Morita, N., and Mikoshiba, K. A. potential approach for gene therapy targeting hepatoma using a liver-specific promoter on a retroviral vector, Cell Struct. Punct., 16: 503-510, 1991), the bone morphogenic protein promoter for brain to target glioma cells (Shirnizu, K. Selective gene therapy of malignant glioma using brain-specific promoters; its efficacy and basic investigation, Nippon Rinsbo, 52: 3053-3058, 1994), the tyrosinase promoter to kill melanoma cells (Vile, R. G., Nelson, J. A., Castleden, S., Chong, H., and Hart, I. R. Systemic gene therapy of murine melanoma using tissue specific expression of the HSVtk gene involves an immune component. Cancer Res., 54:6228-6234, 1994), and the carcinoembryonic antigen promoter for gastric carcinoma cells (Tanaka, T., Kanai. F., Okabe, S., Yoshida, Y., Wakimoto, H., Hamada, H., Shiratori, Y., Lan, K-H., Ishitobi, M., and Omata, M. Adenovirus-mediated prodrug gene therapy for carcinoembryonic antigen-producing human gastric carcinoma cells in vitro. Cancer Res., 46: 1341-1345, 1996). To date, the best studied therapeutic gene is herpes simplex virus TK gene. Herpes simplex virus-TK converts the pro-drug ACV to a phosphorylated form that is cytotoxic to dividing cells (Moolten, F. L., Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes; paradigm for a prospective cancer control strategy. Cancer Res., 46:5276-5281, 1986). Critical to successful results is the xe2x80x9cbystanderxe2x80x9d effect, which confers cytotoxicity on neighboring nontransduced cells; effective tumor cell kill can be achieved without the delivery to and expression of suicide genes in every tumor cell in vivo. This approach has been demonstrated recently to be efficacious in causing regression of many solid tumors, including metastatic colon carcinoma in the rat liver, (Chen, S.II., Chen, X. H. L., Wang, Y., Kosal, K. E., Finegold, J. J., Rich, S. S., and Woo, S. L. C., Combination gene therapy for liver metastasis of colon carcinoma in vivo. Proc. Natl. Acad. Sci. USA. 92:2577-2581, 1995), gastric carcinoma, (Tanaka, T., Kanai. F., Okabe, S., Yoshida, Y., Wakimoto, H., Hamada, H., Shiratori, Y., Lan, K-H., Ishitobi, M., and Omata, M. Adenovirus-mediated prodrug gene therapy for carcinoembryonic antigen-producing human gastric carcinoma cells in vitro. Cancer Res., 46: 1341-1345, 1996), and malignant mesothelioma (Smythe, W. R., Hwang, B. S., Elshami, A. A., Amin, K. M., Eck, S., Davidson, B. L., Wilson, J. M., Kaiser, L. R., and Albelda, S. M. Treatment of experimental human mesothelioma using adenovirus transfer of the herpes simplex thymidine kinase gene. Ann. Surg., 222:78-86, 1995).
Osteocalcin (OC), a noncollagenous Gla protein produced specifically in osteoblasts, is synthesized, secreted, and deposited at the time of bone mineralization (Price, P. A. Vitamin-K dependent formation of bone GLA protein (onteocalcin) and its function. Vitam. Horm., 42:65-108, 1985). A recent study showed that immunohistochemical staining of OC as positive in primary osteoblastic osteosarcoma and chondroblastic osteosarcoma specimens as well as in five of seven fibroblastic osteosarcomas (Park, Y. K., Yung, M. H., Kim, Y. W., and Park, H. R. Osteocalcin expression in primary bone tumors: in situ hybridization and immunohistochemical study. J. Korean Med. Sci., 10:268-273, 1995). In addition, OC activity was detected in a wide spectrum of human tumors. This is consistent with the clinical observations that many human tumors exhibited calcification characteristics both in the primary and at distant metastases.
Because of the poor response rate of previously treated patients with relapsed osteosarcoma to second-line chemotherapy and the fact that many human solid tumors failed to respond to conventional chemotherapy and radiation therapy, it is important to develop new therapeutic approaches that can be applied either separately or in conjunction with current treatment modalities.
Pulmonary metastasis is the main cause of death of patients with several types of cancer, including osteosarcoma, renal cell carcinoma, malignant melanoma, and breast cancer. This application demonstrates the efficacy of the treatment of osteosarcoma pulmonary metastases with a systemic delivery route of Ad-OC-TK supplemented with ACV. We established osteosarcoma lung metastases in nude mice by intravenous injection of rat osteosarcoma cells, ROS 17/2.8. ROS 17/2.8 cells colonized and formed tumor nodules within one week in the lungs of nude mice. Whereas systemic delivery of Ad-RSV-xcex2 Gal(recombinant adenoviral vector containing E. coli beta-galactosidase gene driven by a rous sarcoma virus universal promoter) resulted in non-specific expression of beta-glactosidase (xcex2-gal) activity in the lung parenchyma, Ad-OC-xcex2 Gal administration resulted in specific xcex2-gal expression in tumor cells deposited in the lung. When nude mice bearing ROS 17/2.8 lung tumors were treated with systemic Ad-OC-TK through tail vein administration, subsequent intra peritoneal ACV treatment significantly decreased the number of tumor nodules (p less than 0.0001) and the net lung wet weight (p=0.0005), and increased significantly (0.005 less than p less than 0.01) the survival of animals when compared to untreated and Ad-OC-TK or ACV-treated control groups. These results suggest that Ad-OC-TK plus ACV may be used as a systemic therapy for the treatment of osteosarcoma lung metastasis.
The osteocalcin promoter, and therapeutic agent of this and the parent application, Ad-OC-TK, is not limited to the delivery of therapeutic genes for treatment of tumors. This system is also adapted for the treatment of normal tissue. Co-administration, systemically or locally, of Ad-OC-TK with acyclovir (ACV) may be effective in treating benign prostatic hypertrophy, as well as arteriosclerosis.